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The activation loop tyrosine 823 is essential for the transforming capacity of the c-Kit oncogenic mutant D816V

Abstract

Oncogenic c-Kit mutations have been shown to display ligand-independent receptor activation and cell proliferation. A substitution of aspartate to valine at amino acid 816 (D816V) is one of the most commonly found oncogenic c-Kit mutations and is found in >90% of cases of mastocytosis and less commonly in germ-cell tumors, core-binding factor acute myeloid leukemia and mucosal melanomas. The mechanisms by which this mutation leads to constitutive activation and transformation are not fully understood. Previous studies have shown that the D816V mutation causes a structural change in the activation loop (A-loop), resulting in weaker binding of the A-loop to the juxtamembrane domain. In this paper, we have investigated the role of Y823, the only tyrosine residue in the A-loop, and its role in oncogenic transformation by c-Kit/D816V by introducing the Y823F mutation. Although dispensable for the kinase activity of c-Kit/D816V, the presence of Y823 was crucial for cell proliferation and survival. Furthermore, mutation of Y823 selectively downregulates the Ras/Erk and Akt pathways as well as the phosphorylation of STAT5 and reduces the transforming capacity of the D816V/c-Kit in vitro. We further show that mice injected with cells expressing c-Kit/D816V/Y823F display significantly reduced tumor size as well as tumor weight compared with controls. Finally, microarray analysis, comparing Y823F/D816V cells with cells expressing c-Kit/D816V, demonstrate that mutation of Y823 causes upregulation of proapoptotic genes, whereas genes of survival pathways are downregulated. Thus, phosphorylation of Y823 is not necessary for kinase activation, but essential for the transforming ability of the c-Kit/D816V mutant.

Introduction

c-Kit is a type III receptor tyrosine kinase that is expressed on the surface of hematopoietic progenitor cells, mast cells, germ cells, melanocytes and interstitial cells of Cajal.1, 2 Its physiological ligand stem cell factor (SCF) binds to c-Kit, resulting in dimerization of the receptors and a conformational change comprising release of the auto-inhibitory constraint that the juxtamembrane domain (JMD) poses on the kinase domain, outward opening of the activation loop (A-loop) and sequential phosphorylation of tyrosine residues in the cytoplasmic region of the receptor.3 These events in turn activate the signaling cascades by binding, phosphorylation and activation of various signaling molecules. Oncogenic activating mutations in c-Kit have been described in various tumors in the tissues where c-Kit is normally expressed. 2, 4 The most commonly found mutation in c-Kit is a single-point mutation resulting in the substitution of an aspartate to a valine at position 816 (D816V), which is found in almost all cases of systemic mastocytosis and less commonly in so-called core-binding factor leukemia,5, 6 germ cell tumors and melanomas.4, 7, 8 This mutation results in ligand-independent autoactivation of c-Kit9 which is followed by activation of multiple signaling cascades10, 11, 12, all contributing to aberrant gene expression and tumor progression. This makes D816V a target for small molecule tyrosine kinase inhibitors like dasatinib, PKC 412 and SU-541613, 14, 15, 16, 17, which either directly inhibit D816V or target the downstream effectors controlling mast cell proliferation or cell survival.18 Although a number of such molecules have been in clinical trials, problems with low efficacy, target specificity and resistance to inhibitors remain a major limitation of most inhibitors. Imatinib is a well-known inhibitor of wild-type c-Kit. However, it is not an effective inhibitor of the D816V mutant.19

The A-loop present at the C terminus of the kinase domain spans about 25 amino acids of the c-Kit receptor and is a region known for a number of activating mutations, including the D816V mutation. Upon activation, the A-loop positions itself in the so-called DFG-in state allowing the phosphotransfer from adenosine triphosphate to the tyrosine hydroxyl groups on the receptor. The DFG motif is a conserved tripeptide sequence (DFG) present in the N terminus of the A-loop. In the active kinase conformation, the A-loop extends over the carboxy terminus of the catalytic pocket and the DFG motif moves away from the adenosine triphosphate-binding region (DFG-in), and thereby creates an active conformation of the kinase. Previous studies show that Y823 is crucial for maintaining receptor stability rather than kinase activity.20 It has been also shown that the network via which JMD and A-loop communicate is disturbed by D816V mutation; however, introduction of a D792E mutation in the A-loop restores this interaction pattern.3

In this study, we have investigated the effects of mutating Y823 to phenylalanine (Y823F) on oncogenic c-Kit/D816V signaling. We show that Y823F renders c-Kit/D816V-expressing cells far more sensitive to apoptosis than the cells having Y823 intact. Cell proliferation was also severely reduced in cells expressing the c-Kit/D816V/Y823F compared with cells expressing c-Kit/D816V. Furthermore, the transforming capability of the c-Kit oncogenic mutant was hampered as the cells containing the c-Kit/D816V/Y823F double mutant were unable to phosphorylate STAT5 and lost the ability to form colonies in semi-solid medium. A reduction in phosphorylation of the adaptor proteins Cbl and Shc was also observed. The PI3-kinase/Akt and the Ras/Erk pathways were further perturbed. Tumors formed in mice by Ba/F3 cells expressing the c-Kit/D816V/Y823F double mutant were severely reduced in volume and weight compared with mice injected with c-Kit/D816V expressing cells. Taken together, our data suggest that cells expressing the Y823F mutation can counter balance the uncontrolled proliferation and inhibit apoptosis of c-Kit/D816V-expressing cells.

Results

Y823F mutation does not affect total tyrosine phosphorylation but reduces cell proliferation

We have previously demonstrated that the kinase activity of wild-type c-Kit is unaffected by the Y823F mutation.21 Therefore, we wanted to assess whether mutation of Y823 affects the kinase activity of c-Kit/D816V. To this end, we generated the Y823F mutant in c-Kit/D816V background stably transfected into Ba/F3 cells, as they lack endogenous c-Kit expression (Figure 1a). Cell lines expressing c-Kit/D816V/Y823 and c-Kit/D816V, respectively, were analyzed for phosphorylation of c-Kit. In addition to stably transfected cells, we used transiently transfected COS-1 cells (which similar to Ba/F3 cells also lack endogenous c-Kit expression). The absence of phosphorylation at Y823 was confirmed by western blotting using a pY823-specific antibody (Figure 1b). In agreement with our data on wild-type c-Kit, the Y823F mutation did not impair c-Kit/D816V autophosphorylation (Figure 1b). The intensity of the phosphotyrosine antibody signal was quantitated and normalized for c-Kit expression, further demonstrating that there was no difference in kinase activity owing to the presence of the Y823F mutation (Figure 1c). Taking together, this suggests that Y823 is not involved in the regulation of the kinase activity of c-Kit/D816V.

Figure 1
figure1

Y823F mutation in the c-Kit/D816V oncogenic mutant does not affect phosphorylation of c-Kit receptor but reduces cell proliferation. (a) Ba/F3 cells stably transfected with c-Kit/D816V or c-Kit/D816V/Y823F plasmids, respectively, were labeled with phycoerythrin-conjugated anti-c-Kit antibodies or an isotype control to be analyzed by flow cytometry for cell surface expression. The black peak indicates cells labeled with the isotype control, and the gray peak corresponds to the cells labeled with anti-c-Kit antibody. (b) Ba/F3 cells expressing Ba/F3-c-Kit/D816V and Ba/F3-c-Kit/D816V/Y823F were serum-starved for 4 h at 37 °C. Alternatively, transient transfection was performed in Cos1 cells following overnight starvation at 37 °C. Following starvation, cells were stimulated with SCF for 5 min. Cell lysates were prepared, immunoprecipitated (IP) with anti-c-Kit antibody and analyzed by western blotting. Loss of phosphorylation in Y823F mutant was verified by anti-pY823 antibody. Total receptor phosphorylation was detected using phosphotyrosine (pY) antibody and c-Kit was used as a loading control. (c) Quantification of total phosphorylation was performed by measuring signal intensities from three independent experiments using Multi-Gauge software. GraphPad Prism was used to calculate significance. NS, not significant. Error bars indicate s.e.m. (d) Ba/F3-c-Kit/D816V and Ba/F3-c-Kit/D816V/Y823F cells were grown for 48 h in the presence or absence of SCF and with IL-3. To analyze proliferating cells, EdU was added, and cells were incubated for 2 h at 37 °C. Cells were fixed, labeled with Alexa Fluor 647 and analyzed by flow cytometry.

It has previously been shown that cells expressing the c-Kit/D816V mutant display lower cell surface expression of the receptor than cells expressing wild-type c-Kit.22 It was demonstrated that this was dependent on the kinase activity of c-Kit, as it could be reversed by treatment with a tyrosine kinase inhibitor.23 We could, by flow cytometry analysis, demonstrate that introduction of the Y823F mutation did not alter cell surface expression of c-Kit (Figure 1a). We next wanted to assess whether the Y823F mutation led to any phenotypic changes and analyzed cell proliferation by flow cytometry following EdU incorporation. Proliferation was significantly reduced in cell expressing c-Kit/D816V/Y823F as compared with cells expressing the c-Kit/D816V mutant (Figure 1d). This suggests that while the Y823 site is dispensable for kinase activity, it is involved in signaling downstream of c-Kit/D816V.

Y823 is required for intact Erk and Akt pathway signaling in c-Kit/D816V expressing cells

The D816V mutation induces ligand-independent activation of c-Kit followed by sequential recruitment of several signaling molecules that initiate signaling cascades leading to proliferation, survival and transformation. Despite being constitutively active, the D816V mutant has been reported to only weakly activate Erk and Akt in the absence of SCF24, 25 whereas, in murine myeloid progenitor cells, the D816V mutation renders the regulatory subunit of PI3K constitutively phosphorylated but not Akt and Erk1/2.11 We show that Ba/F3 cells expressing c-Kit/D816V responded to SCF stimulation with strong phosphorylation of Akt, whereas Erk and p38 phosphorylation was constitutive. In the presence of the Y823F mutation, the phosphorylation of Akt as well as Erk were strongly reduced (Figures 2a and b). In contrast, phosphorylation of p38 was constitutive and unaffected by the Y823F mutation (Figures 2a and b). These data suggest that Y823 is involved in signaling downstream of c-Kit/D816V in a selective manner.

Figure 2
figure2

The Y823F mutation in c-Kit/D816V negatively regulates select downstream signaling pathways. Ba/F3-c-Kit/D816V and Ba/F3-c-Kit/D816V/Y823F cells were serum-starved and treated with or without 100 ng/ml SCF. Total cell lysates were separated by SDS–PAGE, electrotransferred to Immobilon P membrane and probed with either phospho-Akt antibody or phospho-Erk1/2 or phospho-p-38 antibodies (a). Membranes were stripped and reprobed with respective non-phosphorylated total protein as loading controls. (b) Signal intensities from three independent experiments were quantified using Multi-Gauge software to calculate the difference in band intensities between the phosphorylated and unphosphorylated protein. GraphPad Prism was used to calculate significance. NS, not significant, **P<0.01, ***P<0.001.

Both cell survival and proliferation are significantly reduced by Y823F mutation

We wanted to ascertain whether the mutation of Y823 in c-Kit had an effect on cell survival. As a complement to the EdU incorporation method, the effect of the Y823F mutation on cell proliferation and survival was investigated by the trypan blue exclusion method. The number of living cells was significantly reduced in cells expressing the c-Kit/D816V/Y823F double mutant (Figure 3a). In addition, the effect of the Y823F mutation on D816V-mediated cell survival was examined by staining the cells with Annexin-V and 7-Amino Actinomycin D and analyzed by flow cytometry. Ba/F3 cells expressing the c-Kit/D816V/Y823F mutant showed >40% reduction in cell survival compared with cells expressing c-Kit/D816V (Figure 3b). These findings are in agreement with the lowered activation of Akt and Erk in the cells expressing c-Kit/D816V/Y823F.

Figure 3
figure3

Cells expressing c-Kit/D816V/Y823F display decreased survival and proliferative capacity compared with cells expressing c-Kit/D816V. Ba/F3-c-Kit/D816V and Ba/F3-c-Kit/D816V/Y823F cells were grown for 48 h in the presence or absence of SCF and IL-3. (a) Viable cells were counted by trypan blue exclusion method (b) cells were also labeled with annexin V and 7-aminoactinomycin D and living cells were analyzed by flow cytometry. IL-3 was used as a positive control. Quantification of labeled cells was performed using FloJo software, and results from three independent experiments were statistically analyzed using GraphPad Prism. ns, not significant, **P<0.01, ***P<0.001.

Y823F mutation abolishes the ability of c-Kit/D816V to form colonies in methylcellulose and reduces the phosphorylation of STAT5

As we observed a marked decrease in both Akt and Erk1/2 phosphorylation in cells expressing c-Kit/D816V/Y823F, we wanted to investigate whether this mutation also affects the growth in semi-solid medium. Ba/F3 cells devoid of growth factors and cytokines were mixed with methylcellulose medium and incubated under humidified conditions for 8 days, whereas c-Kit/D816V was able to form colonies, introduction of the Y823F mutation markedly diminished the ability to form colonies or cell clusters (Figure 4a). This is in agreement with our data demonstrating an increased apoptosis and reduced proliferation of Ba/F3 cells harboring the c-Kit/D816V/Y823F mutant.

Figure 4
figure4

Introduction of the Y823F mutation in c-Kit/D816V leads to the loss of transforming capability. Ba/F3 cells depleted from serum and cytokines were mixed with methylcellulose hematopoietic colony assay medium and incubated at 37 °C in a humidified atmosphere (a) c-Kit/D816V/Y823F mutant cells could not form colonies in semi-solid media, whereas c-Kit/D816V oncogenic mutant retained its colony-formation capability. (b) Stably transfected c-Kit/D816V and c-Kit/D816V/Y823F cells were serum and cytokine starved for 4 h at 37 °C. Cell lysates were prepared, and endogenous STAT5 was immunoprecipitated (IP) with STAT5 antibody. Activation of STAT5 was detected by western blotting using pY antibody. Total STAT5 was used as a loading control. (c) STAT5 phosphorylation versus total STAT5 was quantified using Multi-Gauge software from three independent experiments. GraphPad Prism was further used to calculate the significance. ns, not significant, ***P<0.001.

Signal transducer and activator of transcription (STAT) proteins have been described to transduce signals from the membrane-bound receptors to the cell nucleus through the classical JAK-STAT pathway.26 Activation of STAT proteins has been linked to the expression of genes that are crucial for cell proliferation, differentiation and survival. Whereas STAT1, STAT3 and STAT5 were tyrosine phosphorylated in D816V-transformed cells, only STAT5 was shown to be transcriptionally active in the mast cell line HMG-1.2 that carries the D816V mutation.27 We, therefore, aimed to investigate if phosphorylation of STAT5 was affected by the Y823F mutation. Serum-starved cells were subjected to immunoprecipitation with an anti-STAT5 antibody and phosphorylation was detected by western blotting using a phosphotyrosine antibody. We observed a marked reduction in phosphorylation of STAT5 in cells expressing the c-Kit/D816V/Y823F mutant compared with c-Kit/D816V-expressing cells (Figures 4b and c). This suggests the importance of activated STAT5 in mediating signaling crucial for cell proliferation, survival and probably in the ability of the oncogenic mutant to form colonies in semi-solid media.

The Y823F mutation in c-Kit/D816V leads to a reduction in both tumor weight and volume in athymic mice

To investigate the tumor-forming capacity, five athymic mice were injected with Ba/F3 cells expressing the c-Kit/D816V and c-Kit/D816V/Y823F mutants, respectively. All but one animal developed solid tumors, which were isolated and measured for both weight and volume. Interestingly, one of the mice carrying cells with the Y823F mutation did not develop any detectable tumor. The four other mice carrying the c-Kit/D816V/Y823F mutant cells developed tumors that were significantly smaller as compared with the c-Kit/D816V mutant controls. (Figure 5a). Average tumor weight (Figure 5b) and tumor volume (Figure 5c) were reduced by ~80% in mice injected with cells expressing the c-Kit/D816V/Y823F mutant.

Figure 5
figure5

Introduction of the Y823F mutation in c-Kit/D816V leads to a reduction in both tumor weight and volume in female athymic mice. Five athymic mice (NMRI-Nu/Nu strain) were subcutaneously injected with Ba/F3 cells expressing c-Kit/D816V and c-Kit/D816V/Y823F mutation. (a) Tumors carrying the c-Kit/D816V/Y823F mutant were much smaller in size and volume as compared with tumors developed from cells expressing c-Kit/D816V. Mouse 1 carrying the c-Kit/D816V/Y823F mutant was devoid of any tumor formation. (b, c) Tumors from five mice were excised, weighed and measured 5 days post infection. Statistical analysis was performed using GraphPad Prism, *P<0.05.

Mutation of Y823 leads to upregulation of tumor suppressor genes and downregulation of IL2, IL15, TGFβ1 and Myc responsive genes

As we observed that the Y823F mutation diminishes c-Kit/D816V-mediated cell proliferation, survival and colony formation through weaker phosphorylation of Akt, Erk1/2 and STAT5, we hypothesized that this mutation might influence c-Kit-D816V-mediated gene expression. Therefore, we checked global gene expression using Mouse Gene 2.0 ST Array. Oncogenic c-Kit/D816V initiates aberrant expression of numerous proto-oncogenes and antiapoptotic genes. Cells expressing c-Kit/D816V/Y823F displayed, compared with c-Kit/D816V expressing cells, an upregulation of proapoptotic genes, whereas expression of oncogenes and antiapoptotic genes was suppressed (Figure 6a, Supplementary Table S1 and S2). Furthermore, gene set enrichment analysis suggests that deregulated genes (Supplementary Fig. S1) display enrichment of several signaling pathways (Supplementary Table S3) and oncogenic signatures (Supplementary Table S4). For example, Myc (Figure 6b), IL2 (Figures 6c and e), IL15 (Figure 6d) and TGFβ1 (Figure 6f) pathway genes are downregulated in c-Kit/D816V/Y823F expressing cells. Surprisingly, we also observed that genes that are downregulated under hypoxic conditions are also downregulated in c-Kit/D816V/Y823F-expressing cells (Figure 6g) Thus, we suggest that the Y823F mutation has an opposing role to that of the oncogenic mutation c-Kit/D816V, which is partially mediated through transcriptional initiation of proapoptotic genes as well as suppression of oncogenes and antiapoptotic genes probably by controlling STAT5 activation.

Figure 6
figure6

Cells expressing the c-Kit/D816V/Y823F mutant display downregulated expression of proto-oncogenes and upregulated expression of tumor suppressor genes compared with cells expressing c-Kit/D816V. Total RNA extracted from Ba/F3-c-Kit/D816V and Ba/F3-c-Kit/D816V/Y823F cells were subjected to microarray expression analysis using Affymetrix GeneChip Mouse Gene 2.0 ST Array. (a) Differential gene expression was analyzed and presented using GraphPad Prism. Gene set enrichment analysis shows enrichment in different oncogenic signatures (b-d) and signaling pathways (f-g).

Y823F mutation causes accelerated degradation of c-Kit receptor as compared with c-Kit/D816V

To investigate the possible mechanism by which Y823F mutation exerts its growth inhibitory potential, we performed degradation assay on Ba/F3 cells transfected with c-Kit/D816V and c-Kit/D816V/Y823F. Cycloheximide-treated cells were withdrawn at various time points, lysed and probed with c-Kit antibody. Degradation of receptor having Y823F mutation occurred faster as compared with c-Kit/D816V (Figure 7a). The half-life of c-Kit/D816V/Y823F was only 42 min, as compared with the half-life of c-Kit/D816V, which was calculated as 72 min. (Figure 7b).

Figure 7
figure7

The c-Kit/D816V/Y823F mutant has a higher degradation rate compared with c-Kit/D816V. (a) Ba/F3 cells expressing c-Kit c-Kit/D816V and c-Kit/D816V/Y823F were treated with 100 μM of cycloheximide. Equal amount of cells were withdrawn at different time points followed by lysis and western blotting analysis. (b) Quantification of total c-Kit was performed by measuring signal intensities using Multi-Gauge software. GraphPad Prism was used to calculate half-life.

Discussion

Gain-of-function mutations in tyrosine kinases are a major cause of progression towards transformation.28 The two most commonly found regions of gain-of-function mutations in c-Kit are the JMD and the kinase domain near to the A-loop.29 Together these two regions, located in exon 11 and exon 17, respectively, constitute the mutational hotspots in c-Kit. Whereas ~85% of gastrointestinal stromal tumors result from activating mutation in the JMD, 90% of systemic mastocytosis carry a D816V mutation in the kinase domain. Normally, the JMD maintains the kinase in an auto-inhibitory state and the activation process involves two check points: the release of the JMD from the kinase domain that exposes the catalytic site to the substrate and, second, the A-loop coming to the DFG-in state. Exon 18 of the A-loop of c-Kit is located in the C-lobe of the kinase domain and is a less frequent site for mutation in tumors. To date, rather little is known about the roles of the A-loop tyrosines in other receptors, other than that they are in many cases involved the regulation of kinase activity. Previously, in vitro studies, with recombinant wild-type c-Kit, have demonstrated that the corresponding site, Y823, is dispensable for kinase activity.20 A study from Laine et al.3 suggests that several factors such as binding of a substrate, inhibitor or a point mutation within a protein can perturb the signal propagation and corresponding cellular communication. They further demonstrated that the communication pathway between the JMD and the A-loop is disturbed by the D816V mutation. In the present study, we wanted to investigate the effect of the Y823F mutation in the A-loop on downstream signaling of c-Kit/D816V. We demonstrate that although there is no significant effect on the phosphorylation of c-Kit, the downstream signaling through the Ras/Erk and PI3K/Akt pathways is decreased. Upon ligand stimulation, activation of Akt, as well as of Erk and STAT5, was strongly reduced in cells expressing the c-Kit/D816V/Y823F mutant. In contrast, phosphorylation of p38 remained unchanged, suggesting that the effect on downstream signaling is selective and that the oncogenic mutant of c-Kit partially transduces survival and proliferative signals through the Y823 residue.

We further show that cells expressing the c-Kit/D816V/Y823F mutant have almost a 50% reduction in cell survival and have a significantly lower cell proliferation compared with cells expressing c-Kit/D816V. However, the phenotypic outcome could be altered in the presence of additional mutations owing to a perturbed signaling pathway that maintains communication between distant locations in the c-Kit structure. A recent study demonstrated that the long-distance communication between juxtamembrane and A-loop is disturbed by D816V mutation.3 However, mutating the two major phosphorylation sites in the JMD (Y568, Y570) resulted in the D816V mutant in enhanced proliferation,30 suggesting that alterations in either positive or negative signaling pathways affect the transforming capacity of c-Kit/D816V. Y823 in the A-loop might be involved in binding and activation of signaling molecules whose activation is crucial for the expression of genes required for maintaining the transforming capacity of c-Kit/D816V oncogenic mutant. Our results are in concordance with other previous studies where murine Y821, analogous to c-Kit/Y823, was suggested to be important for cell proliferation and survival.31 Tyrosine residues homologous to Y823 in other receptors, such as epidermal growth factor and platelet-derived growth factor receptor, have also been linked to cell survival and proliferation.32, 33, 34 Given the fact that the effect on signaling is very selective, it is not unlikely that the phosphorylated Y823 forms a binding site for a signal transduction molecule that mediates the described effects. Although most signal transduction molecules have been demonstrated to bind to phosphorylated tyrosine residues outside the kinase domain, there are some exceptions. The corresponding tyrosine in the epidermal growth factor receptor, Y845, was demonstrated to associate with the cytochrome c oxidase subunit II in a phosphorylation dependent manner.32 In the oncogenic fusion protein NPM-ALK, phosphorylated Y343 in the A-loop binds to the protein tyrosine phosphatase SHP1 in a phosphorylation-dependent manner.35 Finally, the adapter protein Grb10 has been demonstrated to associate with the insulin receptor through tyrosine residues in the A-loop.36 We have made attempts to identify any possible interaction partners with phosphorylated Y823 by peptide affinity pull-down, but have so far not been able to identify any selective binding partner (data not shown).

As the Y823F mutation affects cell survival and proliferation, we sought to determine its effect on the transforming capability of c-Kit/D816V. We show that cells expressing the c-Kit/D816V/Y823F mutant lose their ability to form colonies in semi-solid medium. This effect is opposite to the tyrosine mutants of JMD, which instead enhance the transformation potential of D816V.30 We further show that the activation of STAT5 is significantly reduced in cells carrying the c-Kit/D816V/Y823F mutant. Under normal physiological conditions, STAT phosphorylation is tightly regulated but constitutive phosphorylation of STAT proteins has been linked to various human malignancies.37, 38 Studies have shown that c-Kit/D816V can directly phosphorylate several different STAT proteins although in human mastocytoma cell lines only STAT5 is activated as a transcription factor.27 Activated STAT5 has been directly associated with transformation of cells and enhances the aggressiveness of the tumor.39, 40 The exact mechanism of STAT phosphorylation by c-Kit/D816V is not clear at the present time. As STAT5 directly links the receptor to its target genes, the decrease in cell proliferation of cells expressing c-Kit/D816V/Y823F mutation could be causally linked to the decrease in STAT5 activation and thereby downregulate genes that are linked to cell proliferation. The mechanism by which Y823 links to phosphorylation of STAT5 is unclear at present. It could be possible that phosphoylrated Y823 serves to recruit proteins involved in phosphorylation of STAT5, but this remains to be shown. To further verify the influence of the Y823F mutation on tumor formation in vivo, Ba/F3 cells expressing c-Kit/D816V or c-Kit/D816V/Y823F, respectively, were injected into mice. Introduction of the Y823F mutation led both to reduced weight and volume of the tumors. Finally, we show the differences in global gene expression of cells expressing Y823F mutation in comparison with c-Kit/D816V oncogenic mutant using microarrays. The genes upregulated in c-Kit/D816V/Y823F double mutant were mostly tumor suppressor genes. These include Schip1 (Schwannomin-interacting protein 1) and is associated with tumor suppression.41 Annexin 3 is also described as both a tumor suppressor and a tumor-activator protein depending on tumor and cell type.42 Another upregulated gene is Ly75, lymphocyte antigen 75, which is linked to early metastasis in ovarian cancer.43 Inpp5f, which is a polyphosphoinositide phosphatase described in cardiac hypertrophy44, is also upregulated in cells expressing the Y823F mutant. The downregulated genes, however, belong mostly to antiapoptotic pathways or survival pathways. Pim1 and Pim2 are associated with several hematological malignancies and other solid tumors.45, 46, 47 Leukemogenesis through the related receptor mutant, FLT-3-ITD, has been related to increased expression of oncogenic PIM kinases.48 Further, downregulation of Ubiquitin specific proteases, Usp 7 and Usp 18 would enhance receptor degradation, which is in concordance with lower cell survival with Y823F mutation.49 Further, the downregulated genes Myc and Bcl2 are well-characterized oncoproteins.50, 51 Thus, we conclude that tumor suppressor genes and proapoptotic genes are upregulated, whereas genes involved in acute myeloid leukemia pathway in c-Kit/D816V/Y823F-expressing cells are upregulated, which is in concordance with its decreased tumorigenic potential.

We have previously shown that Y823F mutant exhibits accelerated degradation as compared with the wild-type c-Kit receptor.21 We further investigated if this also holds true for Y823F mutation in c-Kit receptor carrying D816V mutation. We observed that c-Kit/D816V/Y823F degrades much faster than c-Kit/D816V and has a half-life of only 42 min as compared with 72 min of c-Kit/D816V receptor. Previous studies have shown that A-loop tyrosines are crucial in maintaining structural stability of the receptor, a mutation of the only potential tyrosine in A-loop of c-Kit receptor, therefore, might destabilize it and cause accelerated and less sustained signaling through the receptor. 20, 21

From previous studies, we know that A-loop tyrosine Y823 is not crucial for kinase activity, and its phosphorylation occurs late during the c-Kit-activation process.20 Our study demonstrates that mutation of Y823 causes aberrant downstream signaling including a reduction in the activation of transcription factor STAT5, which further significantly reduces transforming capacity of the oncogenic D816V mutant. Future studies will aim at identifying the proteins that are likely to bind to phosphorylated Y823 that mediates the effects seen. Given the importance of phosphorylation of Y823 for transformation, it will be of importance to understand the mechanisms by which this phosphorylation is regulated, and compounds that interfere with its phosphorylation could potentially be used as selective antitumor drugs.

Materials and methods

Reagents and antibodies

Transfection reagents used were Lipofectamine 2000 (Life Technologies Europe BV, Stockholm, Sweden) and jetPEI (Polyplus transfections/BioNordika, Stockholm, Sweden). Human recombinant SCF and murine recombinant interleukin-3 were obtained from ProSpec Tany Technogene (Rehovot, Israel). Rabbit polyclonal anti-c-Kit serum and anti-Cbl antibodies have been described elsewhere.52 The phospho-tyrosine antibody 4G10 was bought from Millipore (Solna, Sweden). Antibodies against phospho-p38, p38 and Shc were from BD Transduction Laboratories, Stockholm, Sweden. Anti-phospho-Akt antibody was purchased from Epitomics (Burlingame, CA, USA). Polyclonal anti-Gab2, anti-Akt, anti-phospho-Erk, anti-Erk, anti-STAT5 and horseradish peroxidase-coupled secondary anti-goat antibodies were purchased from Santa Cruz Biotechnology (Dallas, TA, USA). Secondary Horseradish peroxidase-coupled anti-mouse and anti-rabbit antibodies were from Life Technologies.

Cell culture

Ba/F3 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 μg/ml streptomycin, 100 units/ml penicillin and 10 ng/ml recombinant murine interleukin-3. Dulbecco's modified Eagle's medium comprised 10% fetal bovine serum, 100 μg/ml streptomycin and 100 units/ml penicillin and was used to culture COS-1 and EcoPack cells.

Expression constructs

pcDNA3-c-Kit/D816V, pMSCV-c-Kit/D816V constructs were described previously.25 pcDNA3-c-Kit/D816V/Y823F and pMSCV-c-Kit/D816V/Y823F constructs were generated by site-directed mutagenesis using QuikChange mutagenesis XL kit (Agilent Technologies, Stockholm, Sweden). All plasmids were verified by sequencing.

Transient and stable transfection

Transient transfection of COS-1 cells was performed using JetPEI according to the manufacturer’s instructions. Transfected cells were incubated for about 24 h before they were serum-starved overnight. Cells were stimulated with 100 ng/ml SCF for indicated time periods. Cell lysis and immunoprecipitation was performed as described.53 Stable transfections were performed as described.25 Cells expressing c-Kit/D816V or c-Kit/D816V/Y823F were confirmed by flow cytometry.

Immunoprecipitation and western blotting

Stimulated cells were washed 1 × with cold phosphate-buffered saline followed by cell lysis, immunoprecipitation and Western blotting as described elsewhere.53 Immunodetection was performed by enhanced chemiluminescence using horseradish peroxidase substrate (Millipore Corporation, Billerica, MA, USA) and the signals were detected by a CCD camera (LAS-3000, Fujifilm, Tokyo, Japan). Signal intensities were quantified using Multi-Gauge software (Fujifilm).

Cell proliferation and survival assay

Ba/F3 cells were washed three times with RPMI 1640 medium and seeded in 24-well plates (70 000 cells/well). Cells were then incubated either with or without 100 ng/ml SCF or with 10 ng/ml Interleukin-3 for 48 h. Viable cells were counted using trypan blue exclusion method. Cell proliferation was also measured by staining the cells with Click-iT EdU Alexa 647 (Life Technologies) using the manufacturer's protocol. Stained cells were then analyzed by flow cytometry (BD FACSCalibur). Apoptosis was measured using an Annexin-V, 7-amino-actinomycin D (7-AAD) kit (BD Biosciences Pharmingen, Stockholm, Sweden), according to the manufacturer's instructions; double-negative (Annexin-V-/7-AAD-) cells represent viable cells.

Degradation experiment

c-Kit/D816V and c-Kit/D816V/Y823F expressing Ba/F3 cells were incubated with 100 μg/ml of cycloheximide for 1 h and samples were withdrawn at indicated time points. Total cell lysates were subjected to sodium dodecyl sulphate-polyacryl amide gel electrophoresis followed by the detection of c-Kit by western blotting. Antibody against β-actin was used as a loading control. Half-life was calculated using GraphPad prism software.

Colony-formation assay

Ba/F3 cells expressing c-Kit/D816V and c-Kit/D816V/Y823F mutants were cultured in semi-solid methylcellulose medium (MethoCult M3231, Stem Cell Technologies, Grenoble, France) as described elsewhere.25

Animal experiments

Female athymic mice (NMRI-Nu/Nu strain) were used and housed in a controlled environment, and all procedures were approved by the regional ethics committee for animal research (approval no. M69/11). Six million cells in 100 μl Matrigel:phosphate-buffered saline (2.3:1) were subcutaneously injected on the right flank. Mice (n=5 for each group) were monitored daily, and tumors were excised, measured and weighed 5 days after injection. Tumor volume is calculated by (π × l × s2)/6, where l=long side and s=short side.

Gene expression analysis

Ba/F3-c-Kit-D816V and Ba/F3-c-Kit-D816V-Y823F cells were serum- and cytokine- starved for 6 h before extraction of total RNA using RNeasy Mini Kit (Qiagen, Sollentuna, Sweden). Quality of extracted RNA was checked with Bio-analyzer and then subjected to expression analysis using Affymetrix GeneChip Mouse Gene 2.0 ST Array. Raw data were processed for RMA normalization followed by significance analysis of microarrays analysis. In addition, ANOVA analysis was performed. Gene enrichment in signaling pathways was done by Gene Set Enrichment Analysis software (GSEA- Broad Institute). The C2 and C6 data sets were used for gene set enrichment analysis analysis.

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Acknowledgements

This work was supported by grants from the Swedish Research Council, the Swedish Cancer Foundation, Gunnar Nilsson Cancer foundation, the Stiftelsen Olle Engkvist Byggmästare, Royal Physiographic Society in Lund, Ollie och Elof Ericssons Stiftelse and Stiftelsen Lars Hiertas Minne.

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Agarwal, S., Kazi, J., Mohlin, S. et al. The activation loop tyrosine 823 is essential for the transforming capacity of the c-Kit oncogenic mutant D816V. Oncogene 34, 4581–4590 (2015). https://doi.org/10.1038/onc.2014.383

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